Genetically modified cells expressing a TGFbeta inhibitor, the cells being lung cancer cells

ABSTRACT

The present invention relates to compositions comprising a therapeutically effective amount of genetically modified cells containing a genetic construct expressing a TGFβ inhibitor effective to reduce expression of TGFβ, where the genetically modified cells are non-small cell lung cancer (NSCLC) cells or small cell lung cancer (SCLC) cells, and related methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of international applicationnumber PCT/US01/10339, and claims the benefit of priority ofinternational application number PCT/US01/10339, having internationalfiling date of Mar. 30, 2001, designating the United States of Americaand published in English, which claims the benefit of priority of U.S.provisional patent application No. 60/193,497, filed Mar. 31, 2000; bothof which are hereby expressly incorporated by reference in theirentireties.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to compositions comprising atherapeutically effective amount of genetically modified cellscontaining a genetic construct expressing a TGFβ inhibitor effective toreduce expression of TGFβ, where the genetically modified cells arenon-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)cells, and related methods.

[0004] 2. Description of the Related Art

[0005] Lung cancer remains the most prevalent cancer in the westernworld, accounting for 30% of all cancer-related deaths (Ramanathan andBelani, 1997). The current prognosis for patients with lung cancer ispoor. The overall cure rate is estimated as low as 13%. Approximately180,000 new cases of lung cancer are expected in the United States in1999. The majority of these patients will die of their disease with160,000 deaths from lung cancer expected nation-wide in 1999.

[0006] There are two major subdivisions of lung cancer: 1) non-smallcell (NSCLC) and 2) small cell lung cancer (SCLC). Treatment approachesand natural history differ for these two diseases. The majority (80%) ofcases of lung cancer in the United States are NSCLC. Although advancesin the understanding of important clinical and prognostic factors forboth NSCLC and SCLC have been made in the past 20 years, there have beenminimal improvements in therapeutic results. The only curative optionfor patients with NSCLC is local therapy (surgical excision or localirradiation) in patients with early stage disease (I & II) when thetumor is still localized. At diagnosis however, the majority of patientswith NSCLC present with advanced disease, which is not curable bysurgery alone. In advanced stages of disease, systemic chemotherapyand/or irradiation can produce objective responses and palliation ofsymptoms, however, they offer only modest improvements in survival. Themedian survival of patients with non-resectable disease is 6-12 months.Two-year survival rates for stages IIIB and IV NSCLC are 10.8 and 5.4percent respectively. Likewise, five-year survival rates are 3.9 and 1.3percent. Recently, several new drugs have become available for thetreatment of NSCLC including paclitaxel (Taxol), docetaxel (Taxotere),topotecan, irinotecan, vinorelbine, and gemcitabine. While these drugsare improvements over prior chemotherapeutic agents (etoposide,cisplatin and carboplatin), the overall cure rate remains low.

[0007] SCLC is a very aggressive cancer which metastasizes early andoften, and it has a median survival from diagnosis of only two to fourmonths. Localized forms of treatment, such as surgical resection orradiation therapy, rarely produce long-term survival because of thiscancer's propensity for distant metastasis. With chemotherapy, survivalcan be prolonged at least four to five times the media survival rate forpatients who are given no therapy, however the overall survival at fiveyears remains at only 5-10%.

[0008] Since current therapeutic modalities do not significantly enhancelife expectancy in stages of NSCLC or SCLC patients, exploration of newtherapeutic approaches for these patients is justified.

SUMMARY OF THE INVENTION

[0009] Patients bearing tumors of different histologic origin haveelevated levels of Transforming Growt Factors-βs (TGFβs). TGFβs aregrowth factors that are associated with immunosuppression. Suppressio ofthe patients' immune system results in their inability to recognize anddestroy tumors when they firs appear. Furthermore, suppression ofpatients' immunity makes them susceptible to frequent infectionsInjection of genetically engineered tumor cells to block their TGFβproduction makes the gene modifie cells potent vaccines that arerecognized by and can activate the immune system against the tumorActivation of the immune system subsequently causes the recognition andcontrol of the parenta unmodified tumors in the host organisms. Thisphenomenon applies in animal tumor models and in huma clinical trials.Thus, we propose to use this approach in patients with stages ofnon-small cell lung an small cell lung cancers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0010] Lung cancers account for 30% of all death due to cancer in theUnited States (Ramanathan and Belani, 1997). The overall cure rate forlung cancer is 13% and the current prognosis for patients with non-smallcell lung (NSCLC) and small cell lung cancer (SCLC) remains poor.

[0011] It has been documented that patients with progressive tumorgrowth have impaired immune function (Jakowlew et al. 1995, Ransohoff etal 1991; Holladay et al. 1992a; Holladay et al. 1992b). This impairment,commonly characterized as marked immune hyporesponsiveness, is notsolely confined to tumor specific immunity, but rather, is oftenobserved throughout the immune system. Impairment is especially evidentin the cell-mediated or T-cell compartment and is characterized byT-cell lymphopenia and impaired T-cell responsiveness to both tumorspecific and non-tumor specific stimuli (Ransohoff et al. 1991). One waytumors may escape immune surveillance is by expressing lower levels ofMHC Class I and Class II molecules. Other tumors may escape byincreasing the expression of immunosuppressor molecules, such as theTGFβs. It is common to observe tumors utilizing a combination of thesemechanisms.

[0012] Gene therapy has received considerable attention in recent years.Vaccination with tumor cells designed to augment tumor antigenpresentation and induce specific anti-tumor immunity has yieldedpromising but limited results (Holladay et al. 1992). Advances in ourunderstanding of cancer biology and developments in vector technologiesare advancing the therapeutic potential of tumor vaccine approaches. Itis now possible to genetically modify tumor cells for vaccination toexpress specific tumor suppressor genes, immune modulators, drugsensitive genes and antisense gene fragments (Huber et al. 1991; Culveret al. 1992; Trojan et al. 1992; Dranoff et al. 1993; Ram et al. 1993;Trojan et al. 1993; Swisher et al. 1999). In particular preclinical andclinical studies demonstrate the potential of gene therapy approaches intreating lung cancer. Preclinical lung cancer models have shownregression of established tumors and enhanced immunogenicity using anallogeneic lung cancer line genetically modified to express the cytokineGM-CSF and a drug sensitive gene, herpes simplex virus thymidine kinaseadmixed with syngeneic bone-marrow derived dendritic cells (Miller etal. 1998). Preliminary results from phase I clinical trials in patientsusing retroviral gene therapy shows gene therapy to be well toleratedand without toxicity (Swisher et al. 1998).

[0013] Transforming growth factors beta (TGFβ) are a family ofmulti-functional proteins that regulate the growth and function of manynormal and neoplastic cell types (Sporn et al. 1986; Massague 1987;Border and Rouslahti 1992; Jachimczak et al. 1993). They exert a widerange of effects on a variety of cell types and have been shown tostimulate or inhibit cell growth, induce apoptosis and increaseangiogenesis (Merzak et al. 1994; Jennings et al. 1994; Ashley et al.1998a; Ashley et al. 1998b; Jennings et al. 1998). These effects aremediated at the level of signal transduction. TGFβ signal transductionhas been found to affect the expression of more than 20 different genes(Baker and Harland 1997; Heldin et al. 1997; Stiles 1997; Yingling etal. 1997).

[0014] TGFβ exists in three isoforms, known as TGFβ1, TGFβ2, and TGFβ3.Their amino acid sequences display homologies on the order of 70-80%.Human TGFβ proteins and genes encoding them are known in the art.Specifically, TGFβ1 mRNA (GenBank Accession No. XM_(—)008912 andNM_(—)000660), TGFβ2 mRNA (GenBank Accession No. XM_(—)001754 andNM_(—)003238), and TGFβ3 mRNA (GenBank Accession No. XM_(—)007417) fromhuman sources have been documented.

[0015] TGFβ receptor proteins may be type I (55 kDa) or type II (70kDa). TGFβ receptor proteins and genes encoding them are also known inthe art. Human TGFβ receptor type I mRNA (GenBank Accession No.XM_(—)005591) and human TGFβ receptor type II mRNA (GenBank AccessionNo. XM_(—)003094) are described in the art.

[0016] Cytokines of the TGFβ superfamily bind to specificserine/threonine kinase receptors and transmit intracellular signalsthrough Smad proteins. Upon ligand stimulation, Smads move into thenucleus and function as components of transcription complexes. TGFβsignaling is regulated positively and negatively though variousmechanisms. Positive regulation amplifies signals to a level sufficientfor biological activity. Negative regulation occurs at theextracellular, membrane, cytoplasmic and nuclear levels.

[0017] Many tumors, including NSCLC and SCLC, produce high levels ofactive TGFβ (Constam et al. 1992; Eastham et al. 1995; Friedman et al.1995; Jakowlew et al. 1995; Kong et al. 1995; Yamada et al. 1995; Ederet al. 1996). Elevated TGFβ levels have also been linked withimmunosuppression (Sporn et al. 1986; Massague 1987; Bodmer et al. 1989;Border and Rouslahti 1992; Chen et al. 1997). TGFβ inhibits T cellactivation in response to antigen stimulation. Additionally, TGFβ hasantagonistic effects on the Natural Killer (NK) cells as well as theinduction and proliferation of the lymphokine-activated killer (LAK)cells (Rook et al. 1986; Kasid et al. 1988; Tsunawaki et al. 1988; Hirteet al. 1991; Ruffini et al. 1993; Naganuma et al. 1996). In support ofthis, a relationship between TGFβ levels and survival has beendemonstrated in colon cancer (Friedman et al. 1995). Recurrence rateswere 18 fold higher in patients whose tumor produced high levels of TGFβcompared to those whose tumor produced low levels. This relationship wasindependent of nodal status and the degree of differentiation of theprimary tumor.

[0018] Given the role of TGFβ in immune suppression we set out toevaluate the effect of TGFβ inhibition by NSCLC tumor vaccination. Usinga TGFβ inhibitor approach we transfected a number of NSCLC cells withTGFβ antisense, selected from TGFβ1, TGFβ2, and TGFβ3, and mixturesthereof. TGFβ2 antisense was chosen as it demonstrated superiority indownregulating TGFβ expression compared with TGFβ1 or a combination ofTGFβ1 and TGFβ2. These genetically modified NSCLC cells were thenirradiated to prevent proliferation and were injected into a number ofdifferent animal tumor subjects. We observed that NSCLC cells previouslyineffective as a component of a vaccine could be rendered efficaciousthrough such a genetic modification. Blocking TGFβ expression increasedthe immunogenicity of these animals. Furthermore such vaccinationseradicated previously implanted tumors and protected animals from tumorchallenge.

[0019] Given the role of TGFβ in immune suppression we envisionevaluating the effect of TGFβ inhibition by SCLC tumor vaccination.Using a TGFβ inhibitor approach we envision transfecting a number ofSCLC cells with TGFβ antisense, selected from TGFβ1, TGFβ2, and TGFβ3,and mixtures thereof. These genetically modified SCLC cells are thenirradiated to prevent proliferation and are injected into a number ofdifferent animal tumor subjects. We envision observing that SCLC cellspreviously ineffective as a component of a vaccine would be renderedefficacious through such a genetic modification. Blocking TGFβexpression is envisioned as increasing the immunogenicity of theseanimals. Furthermore such vaccinations are envisioned as eradicatingpreviously implanted tumors and protecting animals from tumor challenge.

[0020] We have shown the efficacy of this approach in a NSCLC tumormodel. In the KLN-205 NSCLC tumor model, DB2 mice were vaccinated withtwo injections of 5×10⁵ irradiated TGFβ2 antisense gene modifiedautologous NSCLC cells. This was capable of protecting the animalsagainst a subsequent intraperitoneal (i.p.) tumor challenge with 10⁶unmodified KLN-205 NSCLC cells. In eradication experiments in this lungcancer tumor model, vaccination of animals bearing one week old tumors,with TGFβ2 antisense gene modified cells resulted in marked tumorregression and prolonged tumor free survival compared to the controlgroup.

[0021] Fakhrai et al. 1996 demonstrated the efficacy of this approach ina rat glial tumor. In the 9L gliosarcoma tumor model, intracranialimplantation of as few as 300 tumor cells in Fisher-344 rat resulted inover 99% fatality after six weeks. Fakhrai et al. 1996 implanted 5×10³tumor cells into the brain of rats and administered tumor vaccinations.Animals immunized with TGFβ2 antisense modified 9L cells, or with TGFβ2antisense modified 9L cells genetically modified to secrete IL-2remained tumor free for the duration of the study (24 out of 24 or 100%tumor free survival). In contrast, the majority of the control group (2out of 15) immunized with cells containing the empty vector developedtumors and had to be euthanized within five weeks (13% tumor freesurvival, p<0.01).

[0022] Liau et al. 1998 demonstrated comparable efficacy of TGFβ2antisense gene therapy in a rat C-6 glioma tumor model. Dorigo et al.1998 showed the efficacy of this approach in a murine ovarian teratoma(MOT) tumor model; however, only the group inoculated with TGFβantisense and IL-2 gene modified cells resulted in significantprotection from a subsequent tumor challenge, thus establishing theempiricism of the approach. Other groups have demonstrated similaranti-tumor effects of TGFβ gene therapy in cultured cells and animaltumor models (Kim et al. 1997).

[0023] Gene therapy has received considerable attention in recent years.Vaccination with tumor cells designed to augment tumor antigenpresentation and induce specific anti-tumor immunity has yieldedpromising but limited results. Advances in our understanding of cancerbiology and developments in vector technologies are advancing thetherapeutic potential of tumor vaccination. It is now possible togenetically modify tumor cells for vaccination to express specific tumorsuppressor genes, immune modulators, drug sensitive genes or antisensegene fragments (Huber et al. 1991; Culver et al. 1992; Trojan et al.1992; Dranoff et al. 1993; Ram et al. 1993; Trojan et al. 1993).

[0024] A number of clinical studies have evaluated genetically modifiedallogeneic tumor cell as primary components of immunotherapeutictreatments for brain, skin, colon and breast cancers. The vaccinationregimens have been shown to be safe and to generate humoral and cellularanti-vaccine immune responses. Preliminary results from several phase Iclinical trials using gene therapies in patients with NSCLC have alsodemonstrated the safety of gene therapies approaches for this patientpopulation (Dubinett, 1998; Roth, 1998; Swisher et al. 1998) as well asa SCLC patient population.

[0025] Groups have demonstrated experience in the field of gene therapy,both in a number of animal tumor models and in the clinic. (E.g.,Fakhrai et al. 1995; Sobol et al. 1999.) The FDA has previously approvedat least four INDs that have been submitted investigating gene-modifiedvaccination in patients with cancer:

[0026] Sobol et al., BB-IND # 5812: “Injection of colon carcinomapatients with autologous irradiated tumo cells and fibroblastsgenetically modified to secrete interleukin-2 (IL-2). A Phase I study”

[0027] Sobol et al., BB-IND # 4840: “Active immunotherapy ofglioblastoma with tumor cells or fibroblast genetically modified tosecrete interleukin-2 (IL-2)”

[0028] Sobol et al., BB-IND # 7483: “A Phase I Study of Allogeneic TumorCells Genetically Modified t Express B7.1 (CD80) Mixed with AllogeneicFibroblasts Genetically Modified to Secrete IL-2 i Patients withColorectal Carcinoma”

[0029] Fakhrai et al. BB-IND # 6658: “Proposal for a Phase I ClinicalTrial: A Phase I Study of the Safety o Injecting Malignant GliomaPatients with Irradiated TGFβ2 Antisense Gene-Modified Autologou TumorCells”

[0030] In BB-IND #6658, the FDA approved a Phase

[0031] In IND evaluating TGFβ2 antisense gene therapy in patients withhigh grade glioma. Patients were vaccinated with autologous glioma tumorcells genetically modified with the a TGFβ2 antisense plasmid to blockTGFβ2 expression. Therapy consisted of intradermal injections with5×10⁶, 1×10⁷ or 2×10⁷ cells every 3 weeks for the first 4 months andevery 1-2 months thereafter. To date, 5 patients have been treated.Under the same IND, the FDA approved the compassionate use of apartially haplotype matched allogeneic glioma cell line which was genemodified with the same TGFβ2 antisense vector in a patient withpediatric glioma.

[0032] Overall treatment has been well tolerated with only low grade,transient toxicities reported. No significant adverse reactions at theimmunization sites and no treatment-related abnormalities have beenobserved on monitoring of complete blood counts, serum chemistries andurinalyses. In a few cases transient, mild erythema has been observed atthe injection sites following the second and third subcutaneousinjections with TGFβ2 antisense gene-modified autologous tumor cells.

[0033] Increased levels of CD3+, CD4+ and CD8+ effector cell infiltratesat the injection site and in secondary tumor biopsies have beenobserved. Immune histology of injection site biopsies and tumor obtainedat subsequent operation demonstrates significantly higher number ofimmune infiltrates in comparison to biopsies taken prior to initiationof gene therapy.

[0034] Of the 5 patients treated, 1 patient demonstrated a clinicalresponse, 2 demonstrated enhanced immune response, 1 showed tumorprogression while the fifth patient is still undergoing therapy. In thepatient who had a clinical response, overall MRI scans performed atapproximately 6-week intervals during the first three months oftreatment revealed modest changes in overall tumor size. Waxing andwaning of peri-tumoral edema associated with alterations in Decadrondoses could be observed. However, MRI scans showed tumor regression byseven months with a further improvement in response 3 months later.

[0035] The phase I clinical trial thus demonstrated the safety ofinjecting patients with 5×10⁶, 1×10⁷ or 2×10⁷ of TGFβ2 antisensegene-modified autologous or haplotype-matched tumor cells. Furthermore,it is encouraging to see enhanced immunogenicity and preliminaryclinical responses seen with this vaccination regimen. We contemplatethe application of this gene therapy approach in patients with NSCLC orSCLC.

[0036] The invention encompasses methods and compositions for prolongingthe survival of a subject having a non-small cell lung cancer (NSCLC) ora small cell lung cancer (SCLC) comprising administering to the subjecta therapeutically effective amount of genetically modified cellscontaining a genetic construct expressing a TGFβ inhibitor effective toreduce expression of TGFβ, where the genetically modified cells areNSCLC or SCLCcells. Any method which neutralizes TGFβ or inhibitsexpression of the TGFβ gene (either transcription or translation) can beused to effectuate subject survival. Such approaches can also be usefulfor treatment applications, i.e., to treat NSCLC or SCLC.

[0037] In one embodiment, survival modalities can be designed to reducethe level of endogenous TGFβ gene expression, e.g., using antisense orribozyme approaches to reduce or inhibit translation of TGFβ mRNAtranscripts; triple helix approaches to inhibit transcription of theTGFβ gene; or targeted homologous recombination to inactivate or “knockout” the TGFβ gene or its endogenous promoter.

[0038] Antisense approaches involve the design of oligonucleotides(either DNA or RNA) that are complementary to TGFβ mRNA. The antisenseoligonucleotides will bind to the complementary TGFβ mRNA transcriptsand prevent translation. Absolute complementarity, although preferred,is not required. A sequence “complementary” to a portion of an RNA, asreferred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double-stranded antisense nucleic acids, a single strand of theduplex DNA may thus be tested, or triplex formation may be assayed. Theability to hybridize will depend on both the degree of complementarityand the length of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

[0039] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of TGFβ could be used in anantisense approach to inhibit translation of endogenous TGFβ mRNA.Oligonucleotides complementary to the 5′ untranslated region of the mRNAshould include the complement of the AUG start codon. Antisenseoligonucleotides complementary to mRNA coding regions could also be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′- or coding region of TGFβ mRNA, antisense nucleic acids shouldbe at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 17 nucleotides, atleast 25 nucleotides or at least 50 nucleotides.

[0040] Regardless of the choice of target sequence, it is preferred thatin vitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

[0041] The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors), or agents facilitating transport across the cell membrane(see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. WO88/09810, published Dec. 15, 1988).

[0042] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0043] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0044] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

[0045] Oligonucleotides of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0046] However, it is often difficult to achieve intracellularconcentrations of the antisense sufficient to suppress translation ofendogenous mRNAs. Therefore a convenient approach utilizes a recombinantDNA construct in which the antisense sequence is placed under thecontrol of a strong promoter. The use of such a construct to transfecttarget cells will result in the transcription of sufficient amounts ofsingle stranded RNAs that will form complementary base pairs with theendogenous TGFβ transcripts and thereby prevent translation of the TGFβmRNA. For example, a vector can be introduced such that it is taken upby a cell and directs the transcription of an antisense RNA. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in mammalian cells.Expression of the sequence encoding the antisense RNA can be by anypromoter known in the art to act in mammalian, preferably human cells.Such promoters can be inducible or constitutive. Such promoters includebut are not limited to: the SV40 early promoter region (Bernoist andChambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., 1982, Nature 296:39-42), etc.

[0047] Ribozyme molecules-designed to catalytically cleave TGFβ mRNAtranscripts can also be used to prevent translation of TGFβ mRNA andexpression of TGFβ. (See, e.g., PCT International PublicationWO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy TGFβ mRNAs, the use ofhammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.There are hundreds of potential hammerhead ribozyme cleavage siteswithin the nucleotide sequence of a TGFβ cDNA. Preferably the ribozymeis engineered so that the cleavage recognition site is located near the5′ tend of the TGFβ mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

[0048] The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429433; published International patent-application No. WO 88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in TGFβ.

[0049] As in the antisense approach, the ribozymes can be composed ofmodified oligonucleotides (e.g. for improved stability, targeting, etc.)and need be delivered to target cells which express TGFβ. A convenientmethod of delivery involves using a DNA construct encoding the ribozymeunder the control of a strong promoter so that transfected cells willproduce sufficient quantities of the ribozyme to destroy endogenous TGFβmessages and inhibit translation. Because ribozymes unlike antisensemolecules, are catalytic, a lower intracellular concentration isrequired for efficiency.

[0050] Endogenous TGFβ gene expression can also be reduced byinactivating or “knocking out” the TGFβ gene or its promoter usingtargeted homologous recombination. (E.g., see Smithies et al., 1985,Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompsonet al., 1989 Cell 5:313-321). For example, a mutant, non-functional TGFβ(or a completely unrelated DNA sequence) flanked by DNA homologous tothe endogenous TGFβ gene (either the coding regions or regulatoryregions of the TGFβ gene) can be used, with or without a selectablemarker and/or a negative selectable marker, to transfect target cellsthat express TGFβ. Insertion of the DNA construct, via targetedhomologous recombination, results in inactivation of the TGFβ gene.

[0051] Alternatively, endogenous TGFβ gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the TGFβ gene (i.e., the TGFβ promoter and/or enhancers) toform triple helical structures that prevent transcription of the TGFβgene in target cells. (See generally, Helene, C. 1991, Anticancer DrugDes., 6(6):569-84; Helene, C., et al., 1992, Ann, N.Y. Accad. Sci.,660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15).

[0052] In yet another embodiment of the invention, the activity of TGFβcan be reduced using a “dominant negative” approach to effectuatesubject survival. To this end, genetic constructs which encode defectiveTGFβs can be used to diminish the activity of TGFβ on neighboring cells.For example, nucleotide sequences that direct expression of TGFβs inwhich domains are deleted or mutated can be introduced into targetcells. Alternatively, targeted homologous recombination can be utilizedto introduce such deletions or mutations into the target cell'sendogenous TGFβ gene. The engineered cells will express non-functionalcytokines (i.e., a cytokine that is capable of binding its naturalreceptor, but incapable of signal transduction). Such engineered cellsshould facilitate a diminished response on neighboring cells toendogenous TGFβ ligand, resulting in subject survival.

[0053] In an alternative embodiment, the administration of geneticconstructs encoding soluble peptides, proteins, fusion proteins, orantibodies that bind to and “neutralize” intracellular TGFβ effectuatesubject survival. To this end, genetic constructs encoding peptidescorresponding to domains of the TGFβ receptor, deletion mutants of theTGFβ receptor, or either of these TGFβ receptor domains or mutants fusedto another polypeptide (e.g., an IgFc polypeptide) can be utilized.Alternatively, genetic constructs encoding anti-idiotypic antibodies orFab fragments of antiidiotypic antibodies that mimic the TGFβ receptorand neutralize TGFβ can be used. Such genetic constructs encoding theseTGFβ receptor peptides, proteins, fusion proteins, anti-idiotypicantibodies or Fabs are administered to neutralize TGFβ and effectuatesubject survival.

[0054] Genetic constructs encoding antibodies that specificallyrecognize one or more epitopes of TGFβ, or epitopes of conservedvariants of TGFβ, or peptide fragments of TGFβ are also encompassed bythe invention. Such antibodies include but are not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)2 fragments,fragments produced by a Fab expression library, and epitope-bindingfragments of any of the above. Genetic constructs encoding suchantibodies may be used as a method for the inhibition of TGFβ activityand effectuation of subject survival.

[0055] For the production of antibodies, various host animals may beimmunized by injection with TGFβ, a TGFβ peptide, truncated TGFβ,functional equivalents of TGFβ or mutants of TGFβ. Such host animals mayinclude but are not limited to rabbits, mice, and rats, to name but afew. Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of the immunized animals.

[0056] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

[0057] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

[0058] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adaptedto produce single chain antibodies against TGFβ gene products. Singlechain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0059] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, Fab expression libraries maybe constructed (Huse et al., 1989, Science, 246:1275-1281) to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity.

[0060] Additionally, the enzymes which cleave TGFβ precursors to theactive isoforms may be inhibited in order to block activation of TGFβ.TGFβ must be activated to exhibit its biological effects and enzymes arerequired to cleave the precursor protein. These enzymes may be alteredgenetically to prevent interaction with the precursor protein,preventing cleavage of the protein to its mature form. Transcription ortranslation of these enzymes may be blocked by a means known to the art.These enzymes may alternatively be inhibited by any means known to oneof skill in the art.

[0061] TGFβs bind to serine/threonine kinase receptors and transmitintracellular signals through Smad proteins. Signal transduction may beinterrupted in order to repress signaling initiated by TGFβ. Bydisrupting signal transduction it is possible to prevent theimmunosuppressive effect of TGFβ. This may be accomplished by any meansknown in the art in which the interaction between the TGFβ receptor andthe Smad protein is antagonized or prevented, including administeringgenetically modified cells which express proteins that block or competewith TGFβ receptor and Smad protein interactions. Alternatively, thetranscription or translation of TGFβ receptor or Smad protein may bealtered by any means known in the art in order to prevent signaltransmission along the signaling pathway.

[0062] Target cells genetically engineered to express such a form ofTGFβ inhibitor are administered as an immunogen to patients with NSCLCor SCLC, whereupon they will serve to enhance anti-tumor immuneresponses and thereby prolong survival of tumor-bearing subjects. Suchcells may be obtained from the patient (autologous) or a donor(allogeneic or xenogeneic). For a patient with NSCLC, the geneticallyengineered cells constitute non-small cell lung cancer (NSCLC) cells,which are NSCLC cells by virtue of being derived from a NSCLC ormimicing a NSCLC (i.e., having shared common tumor antigens or epitopeswith a primary NSCLC). Alternatively, for a patient with SCLC, thegenetically engineered cells constitute small cell lung cancer (SCLC)cells, which are SCLC cells by virtue of being derived from a SCLC ormimicing a SCLC (i.e., having shared common tumor antigens or epitopeswith a primary SCLC).

[0063] Autologous cells are cell that are derived from the sameindividual. Allogeneic cells are cells that are derived from anotherindividual of the same species so that the cells have intraspeciesgenetic variations. Xenogeneic cells are cells that are derived from anindividual of a different species so that the cells have interspeciesantigenic differences.

[0064] In one embodiment, an allogeneic (or xenogeneic) NSCLC or SCLCtumor cell line is chosen as the immunogen. Lung tumor cell lines havebeen shown to have shared epitopes with primary tumors (Takenoyama etal. 1998). These investigators showed that MHC class I restricted CTLgenerated against a human lung adenocarcinoma cell line had demonstrablecytotoxicity against another lung tumor cell line. The cross reactivityin these experiments was blocked by anti-MHC class I and anti-CD8monoclonal antibodies, suggesting that shared common tumor antigensexist among lung cancer cells.

[0065] In another embodiment, an allogeneic (or xenogeneic) cellcocktail is used as an immunogen in patients with NSCLC or SCLC. One canemploy more than one, e.g., two, three, four or more cell lines ratherthan one to increase the total number of tumor antigens present.

[0066] In addition, the target cells will have low levels of TGFβexpression owing to transfection with a genetic construct encoding aTGFβ2 inhibitor. Suppression of TGFβ expression by the tumor cells willremove a major source of immune suppression operative at the site ofvaccine injection. A local immune response, directed against theinjected tumor cells will induce a systemic immune response against thepatients' native tumor.

[0067] The target cells are genetically engineered in vitro usingrecombinant DNA techniques to introduce the genetic constructs into thecells, e.g., by transduction (using viral vectors) or transfectionprocedures, including but not limited to the use of plasmids, cosmids,YACs, electroporation, liposomes, etc. The engineered cells can beintroduced into the patient, e.g., in the circulation,intraperitoneally, intradermally, subcutaneously, at the lobes of thelung. Alternatively, the cells can be incorporated into a matrix andimplanted in the body as part of a tissue graft.

[0068] In another embodiment, target cells are engineered to express acoding sequence for one or more cytokines. In one alternative, anexpression vector singly encoding the one or more cytokines isintroduced into the target cells. In another alternative, an expressionvector doubly encoding the one or more cytokines and a TGFβ inhibitor isintroduced into the target cells. In still another alternative, sometarget cells are engineered to express a coding sequence for one or morecytokines and other target cells are genetically modified to express aTGFβ inhibitor. By co-administering the immunostimulatory agent alongwith inhibiting the immunosuppressant TGFβ, a subject's immune responseto tumor cells may be improved. Examples of cytokines useful forpractice of the present invention include interleukin-1, interleukin-2,interleukin-3, interleukin-4, interleukin-5, interleukin-6,interleukin-7, interleukin-8, interleukin-9, interleukin-10,interleukin-11, interleukin-12, interleukin-15, interferon-alpha,interferon-gamma, tumor necrosis factor-alpha, transforming growthfactor-beta, granulocyte macrophage colony stimulating factor, andgranulocyte colony stimulating factor. The level of cytokine expressionshould be regulated such that anti-tumor immunity can be increasedwithout producing significant systemic toxicity in the subject.

[0069] When the target cells to be administered are non-autologouscells, they can be administered using well known techniques whichprevent the development of a host immune response against the introducedcells. For example, the cells may be introduced in an encapsulated formwhich, while allowing for an exchange of components with the immediateextracellular environment, does not allow the introduced cells to berecognized by the host immune system.

[0070] Toxicity and therapeutic efficacy of NSCLC or SCLC cells can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Numbers of NSCLC or SCLC cells which exhibit largetherapeutic indices are preferred.

[0071] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage for humans lies preferably within a range of concentrations thatinclude the ED50 with little or no toxicity. For any number of NSCLC orSCLC cells used in the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a concentration range thatincludes the IC50 (i.e., the concentration of the test material whichachieves a half-maximal inhibition of symptoms) as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

[0072] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in a conventional manner using oneor more physiologically acceptable carriers or excipients. Variousadjuvants may be used to increase the immunological response, includingQS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU,TERamide, PSC97B, Adjumer, PG-026, GSK-1, GcMAF, B-alethine, MPC-026,Adjuvax, CpG ODN, Betafectin, Alum, and MF59 (see Kim et al. Vaccine2000, 18: 597 and references therein). Formulations for injections maybe presented in unit dosage form, e.g., in ampoules or multi-dosecontainers.

[0073] The present invention further provides a therapeutic compositioncomprising the genetically modified cells expressing a TGFβ inhibitorand a therapeutically acceptable carrier. As used herein, atherapeutically acceptable carrier includes any and all solvents,including water, dispersion media, culture from cell media, isotonicagents and the like that are non-toxic to the host. Conveniently, it isan aqueous isotonic buffered solution with a pH of around 7.0. The useof such media and agents in therapeutic compositions is well known inthe art. Except insofar as any conventional media or agent isincompatible with the genetically modified cells of the presentinvention, use of such conventional media or agent in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0074] The therapeutic compositions of the present invention may beadministered to an animal in need thereof. Accordingly, the presentinvention provides methods for inducing an immune response in an animalin need of such response, which comprise administering to an animal animmunologically effective amount of the subject genetically modifedcells. The present invention also provides methods for preventing ortreating a tumor in an animal, which comprise administering to an animalan anti-tumor effective amount of the subject genetically modifiedcells.

[0075] The term “animal” used herein encompasses all mammals, includinghuman. Preferably, the animal of the present invention is a humansubject.

[0076] The immune response induced in the animal by administering thesubject genetically modified cells may include cellular immune responsesmediated primarily by cytotoxic T cells, capable of killing tumor cells,as well as humoral immune responses mediated primarily by helper Tcells, capable of activating B cells thus leading to antibodyproduction. A variety of techniques may be used for analyzing the typeof immune responses induced by the subject genetically modified cells,which are well described in the art; e.g., Coligan et al. CurrentProtocols in Immunology, John Wiley & Sons Inc. (1994).

[0077] The term “preventing a tumor” used herein means the occurrence ofthe tumor is prevented or the onset of the tumor is significantlydelayed. The term “treating a tumor” used herein means that the tumorgrowth is significantly inhibited, which is reflected by, e.g., thetumor volume. Tumor volume may be determined by various knownprocedures, e.g., obtaining two dimensional measurements with a dialcaliper.

[0078] When “an immunologically effective amount”, “an anti-tumoreffective amount”, or “a tumor-inhibiting effective amount” isindicated, the precise amount of the genetically modified cells to beadministered can be determined by a physician with consideration ofindividual differences in age, weight, tumor size, extent of infectionor metastasis, and condition of the patient. It can generally be statedthat a therapeutic composition comprising the subject geneticallymodified cells is conveniently administered in an amount of at leastabout 1 times 10³ to about 5 times 10⁹ cells per dose.

[0079] The administration of the subject therapeutic compositions may becarried out in any convenient manner, including by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation.Conveniently, the genetically modified cells of the present inventionare administered to a patient by subcutaneous (s.c.), intraperitoneal(i.p.), intra-arterial (i.a.), or intravenous (i.v.) injection. Thetherapeutically acceptable carrier should be sterilized by techniquesknown to those skilled in the art.

[0080] The invention is further illustrated by the following specificexample which is not intended in any way to limit the scope of theinvention. We envision substituting TGFβ1 and TGFβ3 for TGFβ2 in thefollowing example. Additionally, we envision substituting SCLC cells forNSCLC cells in the following example.

EXAMPLE

[0081] In a clinical trial we use four human non-small cell lung cancercell lines that have been previously established in tissue culturelaboratory. We gene modify these tumor cells in the laboratory to blocktheir TGFβ secretion. We then use the genetically engineered cells asvaccines in patients with non-small cell lung cancer. Patients areinjected four times, in monthly intervals, with the gene modifiedvaccine cocktails that constitute the four non-self (allogeneic) TGFβantisense gene modified tumor cells. Our rationale for using otherpeople's tumor cells is that lung tumor cell lines belonging todifferent people have been shown to share common characteristics thatare recognized by non-self immune systems. Treated patients areevaluated four months after they enter therapy. Patients that respond totherapy receive an additional four to twelve injections to evaluatewhether their response to therapy can be amplified.

[0082] Patients are randomly assigned to one of three separate cohorts.The vaccine cocktail constitutes an equal number of each of the fourirradiated TGFβ antisense gene modified NSCLC cell lines. The number ofinjected cells in the three cohorts is 1.25×10⁷, 2.5×10⁷, and 5×10⁷cells respectively.

[0083] Response, time to tumor progression, and tumor free survival aremonitored in patients and compared with historical controls and patientsreceiving other forms of therapy. Patients are monitored and evaluatedaccording to standard evaluation criteria of no response, stabledisease, partial response and complete response. The results of thisstudy are used to evaluate the feasibility of additional clinical trialwith TGFβ antisense gene modified tumor cells alone and in combinationwith IL-2 (or other cytokine) gene modification.

[0084] Primary Objective

[0085] The primary objective of the clinical trial is to evaluate theability of increasing doses of a gene-modified tumor cell vaccine toinduce tumor response in patients with NSCLC.

[0086] Study Design

[0087] This study is designed to evaluate the efficacy of immunizationwith increasing doses of an allogeneic tumor cell vaccine in patientswith NSCLC. Patients are followed for clinical response, immunogenicityand safety.

[0088] Eligible patients receive 4 monthly intradermal injections with acell cocktail comprised of equal numbers of four irradiated allogeneicTGFβ2 antisense gene modified NSCLC cell lines. Patients are randomizedto one of the three study cohorts. Patients receive 1.25×10⁷, 2.5×10⁷,or 5×10⁷ gene modified cells respectively.

[0089] When available, tumor samples obtained from study patients at thetime of clinically indicated surgery are used to establish a cell linefor each patient. The patients' tumor cells are then used in precursoranalyses or cytotoxicity assay monitoring of the patients immuneresponses to gene therapy inoculations.

[0090] Vaccine Administration

[0091] Patients receive intradermal injections of the tumor cell vaccineat months 0, 1, 2, 3 and 4. These are administered in an outpatientsetting. The sites of injection are rotated between the upper and lowerextremities. Patients are observed in the clinic for 2 hours followingvaccination. During this observation period, vital signs are taken every30 minutes. Patients experiencing no significant side effects fromtreatment are discharged 2 hours after vaccination.

[0092] Outline of Study Procedures

[0093] Patients are vaccinated according to the schedule outlined in thetable below. Patients are initially treated once a month for 4 months,unless there is documented unmanageable toxicity or clinicallysignificant disease progression requiring intervention with otheranti-cancer therapies. Tumor staging (by comprehensive CT/MRI scans) isperformed at baseline and at weeks 8, 16 and 28 and every 3 monthsthereafter. Patients have serial monitoring of immune response (humoraland T cell responses) every 4 weeks up to week 28 and every 12 weeksthereafter. Patients are monitored closely for toxicity throughout thestudy. In those patients demonstrating benefit from treatment,additional vaccinations, given every 4-8 weeks, are given for up to 12additional vaccinations (total of 16 vaccinations).

[0094] Stopping Rules

[0095] Given that tumor responses from vaccination may follow a periodof initial tumor progression, patients are allowed to stay on study inthe face of non-clinically significant progression at week 8. At week16, such patients must shown no further tumor progression (no more thana 25% increase at week 16 as compared to week 8). Patients whodemonstrate progressive disease at week 16 (compared to week 8) areremoved from study. Overview of Monthly Treatment Schema Day Day Day DayProcedure Screen 1 2 8 29/1 Informed consent X History, Exam¹ X [2] X XPhone contact X Vital signs, weight, PS X [2] X X X Adverse events X [2]X X X Concomitant medications X [2] X X X CBC with differential X [1] XX X Electrolyte panel X [1] X X Metabolism panel X [2] X X Tumorstaging2 X [4] Biopsy of inoculation site X Humoral Immunity³ X [2] XCellular Immunity³ X [2] X Vaccine administration⁴ X X

[0096] Definition of procedures Phone contact: Patients are contacted byphone by the study nurse to assess the degree of inflammation, pain, orpuritis at local injection site CBC with WBC, HCT, HGB, platelet count,% neutrophil, % differential: lymphocytes, % monocytes ElectrolyteSodium, potassium, chloride, carbon dioxide, BUN, panel: creatinine,glucose Metabolism Calcium, phosphorus, AST, ALT, alkaline phosphatase,panel: bilirubin, uric acid, albumin, protein, Tumor staging: Physicalexam, x-rays, CT/MRI as appropriate. All staging should use the samemethod to assess tumor as used at baseline Biopsy of Punch skin biopsyperformed at periphery of inflamed inoculation vaccination site or, ifno inflammation, to include site: vaccination site Humoral Serumanti-tumor titers Immunity: Cellular Immunophenotyping of peripheralblood B-cell and Immunity: T-cell subsets (if sufficient cells areavailable) including CD3, CD4, CD8, CD16, CD20, and CD68. Measure PBCcytokine profile by quantitative (semi- quantitative), NK activity(nonspecific killing), LAK activity (Allo-killing).

[0097] Inclusion Criteria

[0098] Signed informed consent

[0099] >18 years

[0100] Histologically confirmed non-curable NSCLC with measurabledisease and an estimated volume of 125 cc

[0101] Performance status (ECOG)≦2

[0102] Absolute granulocyte count 1,500/mm3

[0103] Platelet count 100,000/mm3

[0104] Total Bilirubin≦2 mg/dL

[0105] AST and ALT≦2×Upper Limit of Normal

[0106] Creatinine≦1.5 mg/dL

[0107] Exclusion Criteria

[0108] Concurrent systemic steroids>20 mg prednisone/day

[0109] Prior splenectomy

[0110] Surgery, chemotherapy, radiotherapy, steroid therapy orimmunotherapy<4 weeks of study entry

[0111] Brain metastases or meningeal lymphomatosis unless treated andstable for 2 months

[0112] Known HIV positive

[0113] Serious non-malignant disease (e.g., congestive heart failure, oractive uncontrolled bacterial, viral, or fungal infections), or otherconditions which, in the opinion of the investigator would compromiseprotocol objectives.

[0114] Prior malignancy (excluding nonmelanoma carcinomas of the skin)unless in remission for≧2 years

[0115] Treatment with an investigational drug within 30 days prior tostudy entry

[0116] History of psychiatric disorder that would impede adherence toprotocol

[0117] Pregnant or nursing women or refusal to practice contraception ifof reproductive potential

[0118] Conduct of the Study

[0119] The study is conducted according to Good Clinical Practice, theDeclaration of Helsinki and US 21 CFR Part 50—Protection of HumanSubjects, and Part 56—Institutional Review Boards. Written, datedinformed consent for the study is obtained from all patients beforeprotocol-specified procedures are carried out. After signing, patientsare given a copy of their informed consent. Approval of this study isobtained from the appropriate Institutional Review Board prior toenrolling patients on study. Consent forms are in a language fullycomprehensible to the prospective patient. Consent is documented eitherby the patient's dated signature or by the signature of an independentwitness who records the patient's consent.

[0120] Tumor Response

[0121] Patients are evaluated by CT/MRI and physical examination.Response is reported using standard outcome measures for clinical trials(complete response (CR), partial response (PR), stable disease (SD) andprogressive disease (PD)). Any response to treatment (either PR or CR)requires two confirmatory staging at least 4 weeks apart.

[0122] Complete response

[0123] Resolution of all measurable disease for a period of at least 4weeks

[0124] Resolution of all evaluable disease for a period of at least 4weeks

[0125] No new lesions (either measurable or evaluable)

[0126] Partial response

[0127] Decrease in the sum of the product of all measurable lesions byat least 50% for a period of at least 4 weeks

[0128] Subjective improvement in evaluable disease for a period of atleast 4 weeks

[0129] No new lesions (either measurable or evaluable)

[0130] Stable disease

[0131] Less than a 50% decrease AND less than a 25% increase in the sumof the products of all measurable lesions

[0132] No new lesions (either measurable or evaluable)

[0133] Progressive disease

[0134] Greater than a 25% increase in the sum of the products of allmeasurable lesions OR new lesions (either measurable or evaluable)

[0135] Evaluation of Immune Response

[0136] Humoral Immune Response Assessment

[0137] Humoral anti-tumor immune responses is evaluated by comparing thetiter of pre-treatment and post-treatment sera for reactivity againstthe vaccinating cell lines using an enzyme-linked immunosorbent assay(ELISA). Briefly, 10⁵ target cells are immobilized on filter paper disksin a 96-well incubator chamber (V and P Enterprises, La Jolla, Calif.)and then incubated for 30 minutes with the test sera. The plates arewashed and then incubated with an enzyme-conjugated anti-human Ig. Theplates are again washed, the enzyme substrate is added, and the bindingis quantitated by measuring the absorbance of each well on an ELISAreader.

[0138] Cellular Immune Response Assessment

[0139] Immunophenotyping

[0140] Standard immunofluorescence flow cytometry assays are performedto assess patients pre and post treatment immune effector cellsprofiles. Percentages of effector cell subpopulations reacting withmonoclonal antibodies to T-cells (CD3, CD4, CD8), natural killer cells(CD16) and B-cells (CD20) are measured in the pre- and post-treatmentperipheral blood lymphocyte population and correlated with patientsresponses measured by other criteria. Briefly, the Ficoll-Hypaquepurified mononuclear cells are incubated with the primary antibody for 1hour at room temperature, washed and then incubated with fluorochromeconjugated secondary antibody. The cells are washed, fixed, and thepercentage of positive cells are determined with a flow cytometer.Incubations of the cells with isotype-matched control antibody insteadof the primary antibody serve as negative controls.

[0141] Natural Killer (NK) Activity

[0142] NK activity is analyzed using a standard chromium release assayusing the NK-sensitive cell line K562 as the target. Briefly, K562 cellsare labeled by incubating them with ⁵¹Cr for 45 minutes at 37° C. Thetarget cells are washed extensively and then 5×10³ K562 are incubatedfor 4 hours at 37° C. with pre- and post-treatment PBMC at effectorcell:target cell ratios ranging from 100:1 to 3:1. The cells arel thencentrifuged and the amount ⁵¹Cr-release is measured using a gammacounter. The percent specific lysis is determined using the formula:(experimental cpm−background cpm)/(total cpm−background cpm)×100.

[0143] LAK Activity

[0144] LAK activity is determined by chromium release assay as describedabove, using the LAK-sensitive cell line DAUDI as the target.

[0145] Pre- and Post-Treatment Cytokine Profile of Lymphocytes

[0146] The cytokine profile of the patients PBMC is determined bysemi-quantitative PCR assays. RNA is extracted from patients pre- andpost-treatment purified mononuclear cells and used to synthesize firststrand cDNA by an Invitrogen (San Diego, Calif.) cDNA cycle kitaccording to the manufacturer's recommendation. The first strand cDNA isthen used as template in PCR assays employing different primer sets fordetection of IL-2, IL-4, IL-6, IL-7, IL-10, GM-CSF, γ-INF, TNF-α, etc.To achieve quantitation PCR reactions are limited to 15-18 cycles. As aninternal control and to aid in quantitation of the products knownconcentrations of a control RNA are added to each sample prior toinitiation of cDNA synthesis. Specific primers for the control sequenceare then added to the PCR reactions. Patient samples cytokine profilesare determined by quantitating patients' PCR products and comparing themwith the control PCR products.

[0147] Skin Biopsy of Immunization Site

[0148] Standard hematoxylin and eosin staining and immunohistochemicalmethods employing monoclonal antibodies to hematopoietic cell subsetsare employed to characterize the immune infiltrates observed in skinbiopsies at immunization sites. Monoclonal antibodies to T-cells (CD3,CD4, CD8), natural killer cells (CD16) and B-cells (CD20) are utilizedfor these studies. Briefly, for the immunohistochemical studies,cryostat sections are fixed in cold acetone and then incubated withprimary antibody for 1 hour at room temperature. The sections are washedand then incubated with horseradish peroxidase conjugated secondaryantibody followed by staining sections with an appropriate chromagensubstrate and examined by light microscopy. Incubations of sections withisotype-matched control antibody instead of the primary antibody serveas negative controls.

[0149] Drug Information

[0150] Clinical Formulation

[0151] The vaccine is provided in frozen vials containing at least20×10⁶ cells per vial.

[0152] Pharmacists Instructions Undiluted material Vial volume: 1 mlAppearance: Cloudy fluid Storage: −176° C. (Liquid Nitrogen). Hazards:Frozen vials are not considered hazardous if unbroken. Vials containcell frozen in a mixture containing 10% dimethyl sulphoxide and 50%fetal calf serum. Handling: Frozen vials are not considered a safetyhazard if unbroken. Broken vials should be disposed of in accordancewith biohazard procedures for cytotoxic drugs. Diluted materialPreparation: Before being injected into patients, a frozen vial isthawed in a biosafety hood and washed twice with serum containing mediumand four times with lactated Ringer's. The cells are then counted andadjusted to the appropriate number of cells per injection in a volume of250-400 μl. The cell suspension is delivered in a capped 1 mL syringe.Drug concentrate: 1.25 × 10⁷, 2.5 × 10⁷, or 5 × 10⁷ cells per injectionin a volume of 250-400 μl. Diluent: Lactated Ringer's Route ofadministration: Intradermal injection

[0153] Storage

[0154] Frozen, unopened vials are stored at −176° C. (Liquid Nitrogen).

[0155] Data Evaluation

[0156] Statistics and Estimated Sample Size

[0157] Patients in the amount of 27-75 are enrolled. This is a two-stagestudy. Each of the three treatment arms initially accrues 9 patients.Should no responses be seen in the first 9 patients, then no furtherpatients are accrued to that treatment arm. If at least 1 response isseen in the first 9 patients, then 16 additional patients are accrued tothat treatment arm for a total of 25 patients per treatment arm.

[0158] Definition of Evaluable Patients

[0159] Patients are considered evaluable for tumor response if they havecompleted at least 2 vaccinations and have undergone the tumor restagingat week 8.

[0160] Patients are considered evaluable for immune response if theyhave had at least 1 vaccination and have had immune analysis at week 4.

[0161] Patients are eligible for toxicity following a singlevaccination.

[0162] Reporting of Outcomes

[0163] Response rates are reported using descriptive statistics andreport rates of CR, PR, SD, and PD in those patient determined to beevaluable. Time to progression following initial therapy is determinedfor those patients experiencing either a CR or PR.

[0164] Secondary endpoints include immune response, duration ofresponse, and safety. These rates are also reported using descriptivestatistics. Safety is reported as percent of patient experiencing agiven adverse event. Mean time to progression for the respondingpopulation is reported using Kaplan-Meier statistics.

[0165] Unmodified NSCLC Cell Lines

[0166] Seven of the eight NSCLC lines used in the production of thisvaccine are established cell lines that are purchased from AmericanTissue Cell Culture (ATCC). The human squamous NSCLC cell line, Rh-2,was established from a surgical resection specimen in the lab of Dr.Steven Dubinett at UCLA in 1994, and is publicly available per Lee etal., J. Immunology 152: 3222, 1994; Huang et al., Cancer Research 55:3847, 1995; Huang et al., J. Immunology 157: 5512, 1996; and Huang etal., Cancer Research 58: 1208, 1998.

[0167] pCHEK/HBA2:TGFβ2 Antisense Plasmid

[0168] The pCHEK vector used to construct the human TGFβ2 antisensecontaining plasmid was derived from the pCEP4 vector (Invitrogen, SanDiego, Calif.). It has been modified slightly to facilitate geneticsubcloning. The resulting carrier vector is pCHEK. Genetic subcloningwas used to insert the TGFβ2 antisense gene fragment (HBA2) into pCHEK.Aliquots of the pCHEK/HBA2 plasmid were examined by restriction enzymeanalyses to ensure 1) the identity of the carrier vector into whichTGFβ2 antisense was cloned and 2) the correct orientation of the TGFβ2antisense insert.

[0169] Test limits: Complete homology with expected DNA fragment sizesafter a series of restriction digests with 14 endonucleases: ApaI,BamHI, BglII, ClaI, EcoRV, HindIII, HpaI, NotI, NruI, PstI, SalI, SacII,ScaI, and XbaI.

[0170] Results: The observed DNA fragments obtained by these restrictiondigests corresponded with the expected fragment sizes of pCHEK/HBA2plasmid. In conclusion, the vector used for subcloning is correct andthe TGFβ2 antisense gene fragment insert is in the correct orientation.The expected fragments of these restriction digests are: ApaI 4604 bp3309 bp 1957 bp 874 bp 219 bp BamHI 10963 bp BglII 10210 bp 753 bp ClaI10963 bp EcoRV 10963 bp HindIII 7540 bp 2787 bp 636 bp HpaI 10251 bp 712bp NotI 10963 bp NruI 5716 bp 5247 bp PstI 7573 bp 1494 bp 1277 bp 619bp SalI 8738 bp 2225 bp SacII 5347 bp 3340 bp 2276 bp ScaI 8021 bp 1915bp 1027 bp XbaI 9579 bp 1384 bp

[0171] TGFβ2 Antisense Insert

[0172] To further ensure the correct sequence and orientation of theTGFβ2 antisense fragment, the insert was tested by sequence analysesusing an ABI-310 Genetic Analyzer (Perkin Elmer, Foster City, Calif.).

[0173] Test limits: Complete homology between plasmid insert and TGFβ2antisense.

[0174] Results: Sequencing results obtained confirmed the presence ofhuman TGFβ2 fragment in the pCHEK vector. These results also confirmedthe correct orientation of the insert. The sequence of the human TGF 2fragment used in construction of the pCHEK/HBA2 vector and its flankingregions in the vector are as follows. Lower case letters represent thetwo vector sequences that flank human TGFβ2 fragment.

[0175] tgtctggatc cggccttgcc ggcctcga (seq id no:2)—vector sequenceflanking the insert—

[0176] Base pair 5 of human TGFβ2AATTCAAGCAGGATACGTTTTTCTGTTGGGCATTGACTAGATTGTTTGCAAAAGTTTCGCATC (SEQ IDNO:1) AAAAACAACAACAACAAAACAAACAACTCTCCTTGATCTATACTTTGAGAATTGTTGATTTCTTTTTTTTATTCTGACTTTTAAAAACAACTTTTTTTTCCACTTTTTTAAAAAATGCACTACTGTGTGCTGAGCGCTTTTCTGATCCTGCATCTGGTCACGGTCGCGCTCAGCCTGTCTACCTGCAGCACACTCGATATGGACCAGTTCATGCGCAAGAGGATCGAGGCGATCCGCGGGCAGATCCTGAGCAAGCTGAAGCTCACCAGTCCCCCAGAAGACTATCCTGAGCCCGAGGAAGTCCCCCCGGAGGTGATTTCCATCTACAACAGCACCAGGGACTTGCTCCAGGAGAAGGCGAGCCGGAGGGCGGCCGCCTGCGAGCGCGAGAGGAGCGACGAAGAGTACTACGCCAAGGAGGTTTACAAAATAGACATGCCGCCCTTCTTCCCCTCCGAAACTGTCTGCCCAGTTGTTACAACACCCTCTGGCTCAGTGGGCAGCTTGTGCTCCAGACAGTCCCAGGTGCTCTGTGGGTACCTTGATGCCATCCCGCCCACTTTCTACAGACCCTACTTCAGAATTGTTCGATTTGACGTCTCAGCAATGGAGAAGAATGCTTCCAATTTGGTGAAAGCAGAGTTCAGAGTCTTTCGTTTGCAGAACCCAAAAGCCAGAGTGCCTGAACAACGGATTGAGCTATATCAGATTCTCAAGTCCAAAGATTTAACATCTCCAACCCAGCGCTACATCGACAGCAAAGTTGTGAAAACAAGAGCAGAAGGCGAATGGCTCTCCTTCGATGTAACTGATGCTGTTCATGAATGGCTTCACCATAAAGACAGGAACCTGGGATTTAAAATA--

[0177] Base pair 935 of human TGFβ2.—agcttgct agcagctggt acccagct (seqid no:3)—vector sequence flanking the insert

[0178] Gene-Modified NSCLC Cell Lines

[0179] Following transfection, clones of each gene-modified cell linewere tested for the presence of pCHEK/HBA2 vector by PCR. Only thoseclones testing positive for pCHEK/HBA2 were chosen for further analysis.

[0180] Results: Colonies from seven of the eight cell lines tested werepositive for TGFβ2 antisense transfection. The cell line NCI-H-292 wasnegative.

[0181] TGFβ2 Downregulation

[0182] Following transfection, clones of each gene-modified cell linewere tested for TGFβ2 downregulation by Enzyme Linked ImmunosorbentAssay (ELISA) and compared to unmodified parental cell lines. Briefly,serum free supernatant of TGFβ2 antisense gene modified cells cultureswas collected after 24 hr and assayed in triplicate for TGFβ2 secretionlevels employing an ELISA kit (Genzyme, Cambridge, Mass.). The humanTGFβ2 was captured by an anti-human TGFβ2 monoclonal antibody andquantitated by reaction with horse radish peroxidase-conjugated goatanti-human TGFβ2 antisera according to the manufacturer'srecommendation. Quantitation was achieved by developing the enzymaticreaction with a chromagen substrate and reading the optical density on amicro-ELISA plate reader. A standard TGFβ2 curve presenting knownconcentrations of TGFβ2 permited quantitation of TGFβ2 secretion by theTGFβ2 antisense gene modified cells.

[0183] Test limits for unmodified tumor cells: Secretion of at least 200pg TGFβ2/10⁶ cells/24 hr.

[0184] Test limits for gene-modified tumor cell lines for vaccine:Lowering of TGFβ2 production by at least 35% relative to unmodifiedparental cells. This test limit has been demonstrated to enhanceimmunogenicity of tumor cells in vaccination regimens, per Lee et al.,J. Immunology 152: 3222, 1994.

[0185] Approximately 3-4 days prior to initiation of therapy, aliquotsof each gene-modified cell line are re-tested for TGFβ2 inhibition. Thesame test limits apply. Gene Unmodified modified TGFβ2 TGFβ2 LevelsLevels % TGFβ2 (ng/10⁶ (ng/10⁶ Down Cell Line Lung Carcinoma cell/24 hr)cell/24 hr) regulation NCI-H-292 Mucoepidermoid ND ND ND NCI-H-460 Largecell 0.67 0.12 82% NCI-H-520 Squamous 2.27 1.43 37% NCI-H-596Adenosquamous 1.5 2.6 −73% NCI-H-661 Large cell 13.1 12.96 1% SK-LU-1Adenocarcinoma 3.58 1.36 62% SK-MES-1 Squamous 1.2 1.36 −13% Rh-2Squamous 1.16 0.1 91%

[0186] The 4 cell lines selected were: NCI-H-460, NCI-H-520, SK-LU-1 andRh-2. In our hands, TGFβ2 antisense gene modifications resulted in37-91% blockage of intrinsic TGFβ2 expression in tumor cells.Suppression of TGFβ2 expression in these cells has been stable forperiod of six to nine months in culture.

[0187] Cell Density

[0188] Prior to patient vaccination cell density is assessed for eachcell line. An aliquot of each cell line to be used is counted using ahemocytometer to ascertain cell density. Equal numbers of eachgene-modified cell line is admixed for the three doses to beinvestigated in this study (1.25×10⁶, 2.5×10⁶ and 5×10⁶ total cellsrespectively).

[0189] Cell Viability

[0190] Prior to patient vaccination cell viability is also assessed foreach cell line. The viability of cells employed for immunization isevaluated by trypan blue exclusion methods. Trypan blue dye is a measureof plasma membrane integrity. Viable cells maintain plasma membraneintegrity and therefore exclude the dye. Dead cells lose membraneintegrity and allow uptake of the dye thus appearing blue. An aliquot ofeach cell line is tested and cells counted in a hemocytometer. Thepercentage of viable “non-blue” cells is determined.

[0191] Test Limits: The viability of the TGFβ2 antisense gene modifiedtumor cells used for therapy must be greater than 50%.

[0192] Clonogenicity

[0193] To ensure safety, all gene-modified tumor cell lines to be usedin patient vaccinations must be irradiated prior to injection. This isto prevent tumor cell growth and replication. Cells are irradiated priorto use with a dose of 10,000 cGy. The selection of this radiation doseis based on discussions with Dr. Herman Suit, Chief of RadiationOncology at Massachusetts General Hospital. This was the lowestradiation dose sufficient to render the tumor cells incapable ofproliferation and tumor formation. It is our desire to utilize thelowest possible radiation dose for the transfected cells to optimize thelevel and duration of TGFβ2 antisense transcription. In addition, wehave tested this irradiation dose in our laboratory on cultured tumorcells of different histologic origins, including human NSCLC, gliomas,colon cancer, and pancreatic carcinoma cell lines and demonstrated thatit is capable of completely arresting colony formation by cultured tumorcells of different histologic origins.

[0194] Samples of unmodified and gene modified human NSCLC cell lineswere irradiated with 10,000 centi-Grays. The irradiated cells were thencultured in T-225 flasks and observed for colony formation. A colony wasdefined as a cluster of 16 growing cells. As presented in the tablebelow, colonies did not form in the irradiated cultures during afour-six week observation period. In contrast, all the non-irradiatedcontrol cultures became confluent after 10-14 days. Cell death occurredapproximately two weeks after initiation of the irradiated cultures.Effect of radiation on primary NSCLC cell cultures Tumor cells RadiationDose (Gys) # Colonies at 5 weeks Control cultures None Confluent after10-14 days NCI-H-292 10,000 None NCI-H-460 10,000 None NCI-H-520 10,000None NCI-H-596 10,000 None NCI-H-661 10,000 None SK-Lu 1 10,000 NoneSK-MES-1 10,000 None Rh-2 10,000 None

[0195] Prior to vaccination, an aliquot of each gene-modified cell lineis thawed and tested for colony formation during a four to six weekculturing period before each lot is deemed safe for patient injection.

[0196] Test Limits: No colony formation.

[0197] Sterility of Cell Lines

[0198] Sterility testing was performed for each unmodified cell line byATCC, the manufacturer of the cells.

[0199] In addition, aliquots of each line were sent to MolecularDiagnostics Associates to assay for the presence of the following viralagents: HIV 1 & 2 HBV CMV HH-6 HCV HTLV EBV Adventitious viruses

[0200] Results: All eight unmodified master cell lines were found to benegative for the presence of bacteria, fungi and viruses.

[0201] During in vitro growth and manipulations each cell line wasroutinely tested for bacterial, fungal, and mycoplasma infection. Toavoid contamination with other cells, cultures were processedindividually at all points during laboratory manipulations. Finally, onthe day of therapy, a sample of the inoculum is retested by a gramstain. Only cells that pass all sterility testing are used for therapy.

[0202] Test limits are: No bacteria, mycoplasma or fungal infections.

[0203] Clinical Grade pCHEK/HBA2 Plasmid

[0204] Following preliminary characterization of pCHEK/HBA2 plasmid, DNAstocks were prepared from bacterial cultures by the alkaline lysismethod of Birnboim and Doly as optimized by Qiagen Corporation (Birnboimand Doly, 1979), and purified on Qiagen EndoFree Giga prep columns. Allsteps were carried out under sterile conditions using ART barrier tipsin the biosafety flow hood. An aliquot of the plasmid DNA was removedand tested for sterility. Briefly 20 μl of plasmid DNA was used toinoculate four culture tubes each containing 5 ml of antibiotic free LB.The cultures were incubated for five days at 37° C.

[0205] Test Limits: No bacteria growth.

[0206] Results: No colonies were observed confirming the sterility ofthe prepared clinical grade DNA.

[0207] Brief General Description of Manufacturing and PackagingProcedures

[0208] Eight established NSCLC cell lines were purchased from AmericanTissue Cell Culture (ATCC) or otherwise and were expanded and frozen asunmodified Master Cell Banks (unMCB). Each line was tested for sterility(bacterial and viral contaminants), clonogenicity and TGFβ2 expression.Aliquots of each line were thawed and transfected with pCHEK/HBA2, avector containing the TGFβ2 antisense transgene, using standardtechniques. Gene-modified cell lines were expanded in culture, underhygromycin selection, to grow sufficient numbers for therapeuticapplications and testing. Expanded cell lines were then assayed fordown-regulation of TGFβ2 expression and sterility. Four NSCLC cell lineswhich demonstrated successful downregulation of TGFβ2 expression andsterility were identified: NCI-H-460, NCI-H-520, SK-LU-1 and Rh-2. Thesecell lines were frozen in aliquots as (1) gene-modified Master CellBanks (gmWCB) and (2) gene-modified Working Cell Banks (gmWCB) andstored in liquid nitrogen. Before use, aliquots of these four cell linesare thawed from the gmWCB, irradiated with 10,000 Gy and re-tested forsterility, clonogeneicity and TGFβ2 downregulation. Only cell lotspassing all test limits are acceptable for vaccine preparation. On theday of injection, sufficient cells from each of the acceptable gmWCBlots are then thawed, irradiated and admixed in equal numbers. Beforepatient vaccination, a sample of the innoculum is tested for bacterialcontamination. If no contamination is detected, vaccination can proceed.

[0209] Tissue Procurement

[0210] The following eight established NSCLC cell lines were obtainedfrom American Type Culture Collection (ATCC) or otherwise, expanded inculture and frozen as the unmodified Master Cell Banks (Total 8 unMCB).Cell lines were cultured in IMDM supplemented with 10% FBS, 25 mM Hepes,2 mM L-glutamine, 1 mM sodium pyruvate, 2.5 μg/ml fungizone, 50 μg/mlgentamycin sulphate, 10⁻⁴ M α-thio-glycerol and non-essential aminoacids. Each cell line was frozen as one lot containing 100 vials at>10⁶cells/vial. Each line was tested for sterility and TGF 2 expression. Thefollowing cell lines were used:

[0211] NCI-H-292

[0212] NCI-H-460

[0213] NCI-H-520

[0214] NCI-H-596

[0215] NCI-H-661

[0216] SK-LU-1

[0217] SK-MES-1

[0218] Rh-2

[0219] Construction of Human TGFβ2 Antisense Expression Plasmid

[0220] PCHEKIHBA2 Plasmid Description

[0221] The pCHEK vector used to construct the human TGFβ2 antisenseexpression plasmid (pCHEK/HBA2) was derived from the pCEP4 vector(Invitrogen, San Diego, Calif.) to facilitate gene modification ofcancer cells and to eliminate safety concerns. The pCHEK vector isidentical to the pCEP4 vector in all regions except the following:

[0222] Kanamycin resistance, instead of the ampicillin resistance isincorporated into the pCHEK vector.

[0223] In the pCHEK vector, a DNA cassette unit consisting of the SV-40early promoter followed by an intron drives the expression of thehygromycin resistance gene. Incorporation of the SV-40 promoter/intronunit is to increase expression of the hygromycin resistance gene tofacilitate selection of gene modified cells in culture.

[0224] The pCHEK/HBA2 plasmid utilizes a CMV promoter to drive theexpression of a 930 base pair human TGFβ2 fragment in antisenseorientation. The TGFβ2 antisense fragment consists of bases 6-935 of the5′ end of the human TGFβ2 cDNA molecule that was ligated in reverseorientation adjacent to and under the control of the CMV promoter. ThepCHEK vector also contains the hygromycin resistance gene driven by theSV-40 early promoter, the Epstein-Barr virus origin of replication, andthe gene for the Epstein-Barr virus nuclear associated protein 1(EBNA-1). Additionally, the vector contains the ColE1 origin andkanamycin resistance genes for selection of bacteria containing vectorduring DNA manufacture. PCHEK VECTOR DOMAINS Fragment containing theDomain domain SV-40 Poly A signal  1-405 Multiple cloning site 406-463CMV promoter  467-1311 TK poly A signal 1312-1843 Hygromycin 1844-2899Intron 2900-3233 SV-40 early promoter 3234-3602 ColE1 Origin & Kanamycinresistance 3603-5188 EBNA-1 5189-7789 Epstein Barr origin of replication(OriP) 7790-1046

[0225] Subcloning TGFβ2 Antisense into pCHEK Vector

[0226] TGFβ2 Isolation

[0227] To construct the pCHEK/HBA2 plasmid we first constructedpCEP4/HBA2, a shuttle vector. Briefly, the plasmid pPC21 (publiclyavailable from Dr. Purchio) containing the human TGFβ2 gene was digestedto completion with EcoRI. The EcoRI ends were blunt ended by adjustingthe reaction conditions to 250 μM each dATP, dCTP, dGTP, and dTTP andadding 3 units Klenow enzyme to initiate the reaction. The reaction wasallowed to proceed for 30 minutes at 37° C. The reaction volume was thenadjusted to 100 μl with TE and phenol/chloroform extracted. The DNA wasisopropanol precipitated and rinsed with 70% ethanol. The 930 base pairTGFβ2 fragment (HBA2) was released from the vector by Hind IIIdigestion. Following electrophoresis in a 1% agarose gel, a gel slicecontaining the 930 bp TGFβ2 fragment was excised and the DNA fragmentextracted by routine oxidized silica (glass powder) method. The TGFβ2DNA was then ready to be ligated into the pCEP4 vector.

[0228] pCEP4/HBA2 Shuttle Vector Construction

[0229] The pCEP4 vector was prepared by digestion with XhoI restrictionenzyme, and blunt ended by Klenow reaction as described above. Followingphenol/chloroform extraction and ethanol precipitation, the vector wasdigested with Hind III and purified by agarose gel electrophoresis/glasspowder method as described above.

[0230] The 930 base pair human TGFβ32 fragment was directionallysubcloned into the pCEP4 vector in antisense orientation. Followingtransformation of E. coli, pCEP4/HBA2 DNA was prepared from severalampicillin resistant transformed bacterial colonies. The isolated DNAfrom these colonies was characterized by restriction enzyme analysis andone clone designated as pCEP4/HBA2 was selected and used forconstruction of the clinical plasmid pCHEK/HBA2.

[0231] pCHEK/HBA2 Expression Plasmid Construction

[0232] Briefly, the pCEP4/HBA2 DNA was digested with restriction enzymesKpn I and Bam HI and the 930 bp TGF 2 antisense insert fragment waspurified by agarose gel electrophoresis. The insert was then ligatedinto the Kpn I and Bam HI digested pCHEK vector, and used to transformbacteria. Following kanamycin selection of overnight culture, pCHEK/HBA2DNA was isolated from several clones and characterized by restrictionenzyme analyses to ensure correct identity. To further ensure thecorrect sequence and orientation of the TGFβ2 antisense fragment, theinsert was tested by sequence analyses using an ABI-310 Genetic Analyzer(Perkin Elmer, Foster City, Calif.).

[0233] Manufacturing of Clinical Grade pCHEK/HBA2 Plasmid

[0234] Following preliminary characterization, one bacterial colonycontaining the pCHEK/HBA2 plasmid was streaked on a Luria-Bertani (LB)agar plate containing 100 μg/ml kanamycin. Following incubation at 37°C., a single bacterial colony was used to inoculate 5 ml of LB brothcontaining 100 μg/ml kanamycin and grown overnight at 37° C. in ashaking bacterial incubator. This overnight culture was used toinoculate 50 ml culture of plasmid containing bacteria. The 50 mlbacterial culture was incubated overnight and used to inoculate flaskscontaining 2 liters of LB plus 100 μg/ml kanamycin. This was grownovernight at 37° C.

[0235] DNA was prepared from bacterial cultures by the alkaline lysismethod of Birnboim and Doly as optimized by Qiagen Corporation (Birnboimand Doly, 1979), and purified on Qiagen EndoFree Giga prep columns. Allsteps were carried out under sterile conditions using ART barrier tipsin the biosafety flow hood.

[0236] An aliquot of DNA was removed for restriction analysis anddetermination of DNA concentration. The plasmid DNA concentration wasadjusted to one mg/ml, divided into aliquots, and stored at −70° C. forfuture use. An aliquot of the plasmid DNA was removed and tested forsterility.

[0237] Genetic Modification of NSCLC Tumor Cell Lines with pCHEK/HBA2Plasmid

[0238] Four aliquots of each NSCLC cell line were removed from theappropriate unMCB and grown in culture. Cells were fed with fresh mediumtwice a week. On the day of gene modification cells were fed with freshmedium. Four hours later cells were trypsinized, washed with serumcontaining medium and subsequently PBS. Cell density was adjusted to1-2×10⁷ cells per ml in a volume of 350 μl PBS and incubated on ice for15-20 minutes. 50 μg pCHEK/HBA2 plasmid was added. The mixture wasincubated on ice for an additional 10-15 minutes. The cell suspensionwas then transferred to a pre-chilled cuvette and incubated on ice.After five minutes the cuvettes were placed in a square waveelectroporator (Genetronics, San Diego, Calif.) and subjected to threeelectroporation pulses of 3000 v/cm each pulse lasting 75 μseconds. 1 mlof cold fresh media contining 50 mM Hepes was added and the mixtureincubated at room temperature for 10 minutes. Cells were then plated for2 division cycles and then selection was begun (72 hours aftertransfection). Fresh medium containing 25 μg hygromycin/ml was added tothe cultures. The gene-modified cells were expanded in culture andfrozen as gene modified Master Cell Banks (gmMCB) for a total of 4gmMCBs per cell line. The presence of pCHEK/HBA2 plasmid in genemodified cells was ascertained by PCR. In addition, five vials (5%) fromeach gmMCB were submitted and used for sterility testing consisting ofaerobic, anaerobic, mycoplasma, and fugal assays.

[0239] Preparation and Identification of Cell Lines for PatientVaccination

[0240] Following genetic modification, tumor cell lines were expanded inculture to grow sufficient cells for therapeutic applications andtesting. Clones from each cell line passing identity, strength andsafety test limits were cryopreserved in liquid nitrogen as aliquots ofgene-modified Master Cell Banks (gmMCB) and gene-modified Working CellBanks (gmWCB). Prior to patient vaccination, aliqouts from each gmWCBlot are thawed and irradiated with a dose of 10,000 cGy, a radiationdose we have demonstrated to be capable of completely arresting colonyformation by cultured tumor cells of different histologic originsincluding these NSCLC cells. Aliqouts from each lot undergo safety,strength and identity tests to ensure alteration or contamination hasnot occurred during cell manipulations and freezing. Lots are testedfor: clonogenicity, sterility and TGFβ2 downregulation.

[0241] Cell Preparation and Testing Prior to Patient Vaccination

[0242] Prior to subcutaneous immunization, aliquots of the four chosencell lines are placed in short term cultures. Gene-modified cells aredetached from culture dishes, washed and resuspended in medium,irradiated with 10,000 cGy, washed and resuspended in lactated Ringer'ssolution. They are then tested for sterility and viability. Only cellspassing test limits are used for patient vaccination.

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[0294] While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference.

1 3 1 927 DNA Artificial Sequence Human Transforming Growth FactorBeta-2 1 aattcaagca ggatacgttt ttctgttggg cattgactag attgtttgcaaaagtttcgc 60 atcaaaaaca acaacaacaa aacaaacaac tctccttgat ctatactttgagaattgttg 120 atttcttttt tttattctga cttttaaaaa caactttttt ttccacttttttaaaaaatg 180 cactactgtg tgctgagcgc ttttctgatc ctgcatctgg tcacggtcgcgctcagcctg 240 tctacctgca gcacactcga tatggaccag ttcatgcgca agaggatcgaggcgatccgc 300 gggcagatcc tgagcaagct gaagctcacc agtcccccag aagactatcctgagcccgag 360 gaagtccccc cggaggtgat ttccatctac aacagcacca gggacttgctccaggagaag 420 gcgagccgga gggcggccgc ctgcgagcgc gagaggagcg acgaagagtactacgccaag 480 gaggtttaca aaatagacat gccgcccttc ttcccctccg aaactgtctgcccagttgtt 540 acaacaccct ctggctcagt gggcagcttg tgctccagac agtcccaggtgctctgtggg 600 taccttgatg ccatcccgcc cactttctac agaccctact tcagaattgttcgatttgac 660 gtctcagcaa tggagaagaa tgcttccaat ttggtgaaag cagagttcagagtctttcgt 720 ttgcagaacc caaaagccag agtgcctgaa caacggattg agctatatcagattctcaag 780 tccaaagatt taacatctcc aacccagcgc tacatcgaca gcaaagttgtgaaaacaaga 840 gcagaaggcg aatggctctc cttcgatgta actgatgctg ttcatgaatggcttcaccat 900 aaagacagga acctgggatt taaaata 927 2 28 DNA ArtificialSequence Human Transforming Growth Factor Beta-2 2 tgtctggatc cggccttgccggcctcga 28 3 26 DNA Artificial Sequence Human Transforming GrowthFactor Beta-2 3 agcttgctag cagctggtac ccagct 26

What is claimed is:
 1. A composition comprising a therapeuticallyeffective amount of genetically modified cells containing a geneticconstruct expressing a TGFβ inhibitor effective to reduce expression ofTGFβ, wherein said genetically modified cells are lung cancer cells. 2.The composition of claim 1, wherein said lung cancer cells are non-smallcell lung cancer (NSCLC) cells.
 3. The composition of claim 1, whereinsaid lung cancer cells are small cell lung cancer (SCLC) cells.
 4. Thecomposition of claim 1, wherein said TGFβ is TGFβ-1.
 5. The compositionof claim 1, wherein said TGFβ is TGFβ-2.
 6. The composition of claim 1,wherein said TGFβ is TGFβ-3.
 7. The composition of claim 1, wherein saidgenetically modified cells are autologous cells.
 8. The composition ofclaim 1, wherein said genetically modified cells are allogeneic cells.9. The composition of claim 1, wherein said genetically modified cellsare mixtures of autologous and allogeneic cells.
 10. The composition ofclaim 1, wherein said genetically modified cells further express one ormore cytokines having immunostimulatory effects.
 11. The composition ofclaim 10, wherein said one or more cytokines is selected from the groupconsisting of interleukin-1, interleukin-2, interleukin-3,interleukin-4, interleukin-5, interleukin-6, interleukin-7,interleukin-8, interleukin-9, interleukin-10, interleukin-11,interleukin-12, interleukin-15, interferon-alpha, interferon-gamma,tumor necrosis factor-alpha, transforming growth factor-beta,granulocyte macrophage colony stimulating factor, and granulocyte colonystimulating factor.
 12. The composition of claim 1, wherein said TGFβinhibitor is an antisense inhibitor.
 13. The composition of claim 1,wherein said TGFβ inhibitor is an antisense inhibitor comprising thesequence of SEQ ID NO:1.
 14. The composition of claim 1, wherein saidTGFβ inhibitor is a ribozyme.
 15. The composition of claim 1, whereinsaid TGFβ inhibitor is a dominant negative mutant.
 16. The compositionof claim 1, wherein said TGFβ inhibitor is an antibody.
 17. Thecomposition of claim 1, wherein said TGFβ inhibitor prevents TGFβreceptor and Smad protein interaction.
 18. The composition of claim 1,wherein said composition is provided in unit dosage form.
 19. A methodfor prolonging survival of a subject having a lung cancer comprising thestep of administering to said subject a composition comprising atherapeutically effective amount of genetically modified cellscontaining a genetic construct expressing a TGFβ inhibitor effective toreduce expression of TGFβ, wherein said genetically modified cells arelung cancer cells.
 20. The method of claim 19, wherein said lung cancercells are non-small cell lung cancer (NSCLC) cells.
 21. The method ofclaim 19, wherein said lung cancer cells are small cell lung cancer(SCLC) cells.
 22. The method of claim 19, wherein said TGFβ is TGFβ-1.23. The method of claim 19, wherein said TGFβ is TGFβ-2.
 24. The methodof claim 19, wherein said TGFβ is TGFβ-3.
 25. The method of claim 19,wherein said genetically modified cells are autologous cells.
 26. Themethod of claim 19, wherein said genetically modified cells areallogeneic cells.
 27. The method of claim 19, wherein said geneticallymodified cells are mixtures of autologous and allogeneic cells.
 28. Themethod of claim 19, wherein said genetically modified cells furtherexpress one or more cytokines having immunostimulatory effects.
 29. Themethod of claim 28, wherein said one or more cytokines is selected fromthe group consisting of interleukin-1, interleukin-2, interleukin-3,interleukin-4, interleukin-5, interleukin-6, interleukin-7,interleukin-8, interleukin-9, interleukin-10, interleukin-11,interleukin-12, interleukin-15, interferon-alpha, interferon-gamma,tumor necrosis factor-alpha, transforming growth factor-beta,granulocyte macrophage colony stimulating factor, and granulocyte colonystimulating factor.
 30. The method of claim 19, wherein said TGFβinhibitor is an antisense inhibitor.
 31. The method of claim 19, whereinsaid TGFβ inhibitor is an antisense inhibitor comprising the sequence ofSEQ ID NO:1.
 32. The method of claim 19, wherein said TGFβ inhibitor isa ribozyme.
 33. The method of claim 19, wherein said TGFβ inhibitor is adominant negative mutant.
 34. The method of claim 19, wherein said TGFβinhibitor is an antibody.
 35. The method of claim 19, wherein said TGFβinhibitor prevents TGFβ receptor and Smad protein interaction.
 36. Themethod of claim 19, wherein said composition is provided in unit dosageform.